Process for the enumeration and identification of microorganisms

ABSTRACT

A test for the detection, enumeration and identification of microorganisms in a fluid sample is taught. The test uses a filter through which a fluid sample is passed and onto which organisms within the fluid are deposited. It is then incubated on a growth medium and thereafter subjected to a presence absence test for organisms using an ATP/bioluminescence test. The same sample and filter is then subjected to a PNA probe assay for targeted organisms. Optionally, the locations of the organisms detected by each test can be compared with each other to determine the possibility of false positives and their elimination from the test results.

The present invention relates to a process for enumerating and thenidentifying microorganisms. More particularly, it relates to a processfor enumerating and identifying microorganisms on a membrane device.

BACKGROUND OF THE INVENTION

The traditional method of determining the presence or absence ofmicroorganisms in liquids such as water, food products such as juices,or pharmaceuticals has been to filter the fluid through a suitablemembrane to trap any microorganisms present in the fluid on the surfaceof the membrane. The membrane is then placed on a growth media platesuch as a Petri Dish filled with agar or other suitable media and thenincubated for several days to allow colonies to develop from thecaptured organisms. The plates are then removed and examined visually sothat the number of colonies present can be counted. If the number ishigh enough further tests can be conducted to determine exactly whatorganisms are present. (Often the mere presence of organisms is not inand of itself an indication that the fluid is unsafe and further work toidentify the specific organisms is required.).

Recently, molecular tests such as DNA arrays, nucleic acid amplificationand nucleic acid sequencing have been introduced. Traditionalidentification is done by using selective media and a variety ofbiochemical tests.

It is known that the simple enumeration of such organisms can beexpedited using an ATP-bioluminescence test, such as is taught by U.S.Pat. No. 5,627,042.

It has also been suggested that one may use PNA probe assays to detectthe presence and number of specific microorganisms. See U.S. Pat. No.5,773,571. One drawback to the use of this system is the presence ofspots that do not arise from the presence of microorganisms (falsepositives). Something, yet undetermined, causes the generation of asignal indicating the presence of a target organism when furtherinvestigation reveals that in fact no organism is present in thatlocation. These are probably caused by nonspecific binding of probemolecules to the membranes. This renders the test by itself unreliable,as often the detection of even one target organism is enough to fail thefluid being tested.

Further, none of these tests have been able to detect the presence oflow levels of contaminating organisms in large volumes of fluids, andrapidly identify the organisms of concern with the same sample and inrelatively short time.

What is needed is a simple process to combine the best of both processeswith the same sample to determine the number of total organisms presentas well as the number of those that are of concern.

The present invention provides such a process.

SUMMARY OF THE INVENTION

The present invention relates to a process for the enumeration andidentification of specific species of microorganisms in large volumes offluid using first an enumeration test such as an ATP-bioluminescenceassay or fluorescence indication test to enumerate the total number oforganisms and then a PNA probe assay or assays to determine the presenceof selected or targeted organisms. A key attribute of the presentprocess is that the same membrane and organism sample is used for bothtests. This allows for the rapid detection and identification oforganisms that are present by only making and incubating one sample forboth tests. Moreover, by using the same sample and membrane for bothtests, one can eliminate any false positives by comparing the locationsof the organisms indicated by each test relative to one or moreregistration marks and eliminating those that fail to appear at the samelocation in both tests.

It is an object of the present invention to provide a process forenumerating and identifying microorganisms comprising:

a) filtering a liquid sample through a membrane suitable for theretention of microorganisms,

b) incubating the membrane with any microorganisms,

c) applying an enumeration test to the surface of the membrane toenumerate the microorganisms which are present, and counting the numberpresent,

d) applying a PNA hybridization to determine the presence/absence of oneor more specified microorganisms, and counting the number detected.

It is another object of the present invention to provide a process forenumerating and identifying microorganisms comprising:

(a) filtering a liquid sample through a membrane suitable for theretention of microorganisms,

(b) incubating the membrane with any microorganisms,

(c) applying an enumeration test to the surface of the membrane toenumerate the microorganisms which are present, and counting the numberpresent,

(d) applying a PNA hybridization to determine the presence/absence ofone or more specified microorganisms, and counting the number detected,and

(e) comparing the location of the organisms detected by (c) with thosedetected by (d) and eliminating from the analysis those which are notfound in the same location by each test.

It is a further object of the present invention to provide a process forenumerating and identifying microorganisms comprising:

(a) filtering a liquid sample through a membrane suitable for theretention of microorganisms and which has a registration mark toindicate a specific location on the membrane,

(b) incubating the membrane with any microorganisms,

(c) applying an enumeration test to the surface of the membrane toenumerate the microorganisms that are present, and counting the numberpresent,

(d) applying a PNA hybridization to determine the presence/absence ofone or more specified microorganisms, and counting the number detected,and

(e) comparing the location of the organisms detected by (c) with thosedetected by (d) by comparison to the registration mark and eliminatingfrom the analysis those which are not found in the same location by eachtest.

IN THE DRAWINGS

FIG. 1 shows a block diagram of a preferred embodiment of the presentinvention.

FIG. 2 shows a block diagram of a second preferred embodiment of thepresent invention.

DETAILED DESCRIPTION OF THE SPECIFICATION

The present invention relates to a process for the rapid enumeration andidentification of microorganisms in fluids or soluble solids such aspowders, salts, creams, tablets, etc. Such fluids include but are notlimited to water, potable or otherwise, dairy such as milk, beer, wine,soft drinks, fruit juices, pharmaceuticals, parenterals, bacterial aircounts, etc.

FIG. 1 shows a first preferred process according to the presentinvention. In this process, as the first step 1, a sample in fluid formis obtained from the product to be diagnosed. For example, inenvironmental sampling, such as a water supply, a sample of water fromthe river, well or reservoir is obtained. For a food or pharmaceuticalproduct, a sample is taken from the stock (typically this is thefinished stock although it need not be so). If in liquid form it issimply used as is or may if necessary due to viscosity be diluted withdeionized water. If in solid form, it is dissolved or dispersed in asuitable liquid, typically water or a buffer or other such media thatdoesn't kill or unduly stress organisms that are present.

It is then filtered through a membrane filter in the second step 2 andthe filter is then placed on to a growth medium such as agar (typicallycontained in a Petri Dish) and incubated as the third step 3 to allowmicrocolonies (visible using ATP bioluminescence and imaging, but notvisible by eye) to arise from the trapped organisms.

A preferred device for holding the filter is a MILLIFLEX™ filter funnelhaving a 50 mm diameter and a 100 ml capacity, available from MilliporeCorporation of Bedford, Mass. Other membrane holders such as glass orstainless steel filter holders or funnels may be used instead. Suchdevices are well known and available from a variety of sources includingMillipore Corporation of Bedford, Mass. and Fisher Scientific, Inc, ofPittsburgh, Pa. A preferred membrane is a MicroStar™ filter, availablefrom Millipore Corporation of Bedford, Mass. This filter is a 0.45nominal pore size PVDF filter having a series of hydrophiliccompartments separated by hydrophobic partitions that extend through theentire depth of the filter. Other filters that are useful in thisinvention include EZ-Pak™ filters, S-Pak™ filters, S-Pak™ Plus filtersand AO™ or SO™ filters, available from Millipore Corporation of Bedford,Mass.

The filter/growth medium is incubated in the third step 3 for a shortperiod of time, typically from 1 to about 24 hours. The length ofincubation depends upon the accuracy needed by the test, the rate ofgrowth of the organisms to be detected, the stress to which theorganisms have been subjected (the more stress imposed on an organism,the more incubation time is necessary), the type of organism to bedetected and the sensitivity of the test used to determine the presenceof that organism (the less sensitive the test, the longer the incubationtime to ensure that the colony of the target organism has grown to asuitable size), the desired speed of the test vs. the accuracy of thetest results and other such factors. The temperature for incubation canbe from about room temperature (24° C.) to about 40° C. Higher or lowertemperatures may be used depending upon the organism(s) that one wishesto detect and their optimum temperatures for incubation.

It should be understood that the assays run herein with both theenumeration test and PNA test are on microcolonies of microorganismsthat develop from the single cells captured on the membranes i.e. one isenumerating the numbers of single organisms in a test sample bydetection of colonies that arise by growth division from those samesingle organisms rather than detecting the individual organismsdirectly.

After a suitable incubation period for the filter on the medium, thefilter is removed from the media, dried, then sprayed with reagents, andsubjected to an enumeration test 4.

A preferred enumeration test is an ATP/bioluminescence test. In such atest, the sample is exposed to a suitable permeabilizing agent such asATP releasing agent from the MicroStar reagent kit (RMLL00000) availablefrom Millipore Corporation of Bedford, Mass. ATP is then detectedthrough the use of a bioluminescence reagent such as BioluminescenceReagent from the MicroStar reagent kit available from MilliporeCorporation of Bedford, Mass. The luminescence is then read visually ordigitally through an imagining device such as CCD camera with imageprocessor. One such system is sold as MicroStar™ system, available fromMillipore Corporation of Bedford, Mass. By whatever means that is used,the count and location of the microbial colonies detected can be made.

Alternatively, one may use the process of U.S. Pat. No. 5,627,042 theteachings of which are incorporated herein by its entirety. In thisprocess, ATP is extracted from a living organism on a membrane throughthe use of an extraction agent such as NRB™ available from Lumac, Co.and then subjected to a bioluminescence agent such as Lumit-PM™ fromLumac Co. The luminescence is then measured on a device that detects andrecords the location of the light emitted by the ATP/reagent reaction.However, this process is not preferred as it actually ruptures the cellwall spilling its contents on to the membrane surface. Even though themembrane has hydrophobic partitions, it can not be guaranteed that somecross contamination will not.

A further alternative is to use a system for enumeration that does notrely on the detection of ATP but uses some other mechanism to determinethe presence and the number of organisms. One such system is ScanRDIavailable from Chemunex. Rather than using ATP, it uses a fluorescenttag that is absorbed by the organisms (fluorescent tag absorption test)and then detected under a fluorescent light.

After the enumeration test, the sample is then subjected to a PNA probeassay 5 for the target organisms. PNA probes are peptide nucleic acidprobes (rather than DNA based probe) and are designed to enter a celland hybridize to a select portion of an RNA or DNA strand of that cell.They may be designed so as to specifically hybridize only to a singlespecies or to an entire genus. The probes contain a tag such as anenzyme, hapten, fluorophore or radioisotope to indicate their presencein the cell. Such probes are available from a variety of sourcesincluding Boston Probes, Incorporated of Bedford, Mass. under tradenamessuch as Eco06™ probes for the detection of E. coli and BacUni™ probesfor the simple detection of all bacteria. The method of making and usingsuch probes are taught by U.S. Pat. Nos. 5,714,331; 5,736,336; 5,773,571; 5,912,145 and 5,985,563; and PCT published applications WO 98/24933and 99/49293, the teachings of which are hereby incorporated in theirentireties.

In this step, the organisms on the same filter are subjected again to apermeabilizing and fixation agents such as ethanol and glutaraldehyde,and PNA probes are applied to the permeable cells. One may select one ormore PNA probes to detect one or more species of organisms. Some existfor individual organisms such as E. coli or Salmonella sp., L. brevis,etc, while others are more universal and simply detect the presence ofbacteria or yeast or fungi. After application of the probe(s), themembrane is allowed to sit for a period of time, typically 10 minutes to1 hour and is then washed to remove any unhybridized probes. Dependingupon probes selected and the method used to incorporate a detectable tagon it, one can use X-ray film, instant film, fluorescence, colorimeteror other such device to detect the presence of the tag of the probeswhich have hybridized to the DNA or RNA of the organism and therebyidentify the number and type of each target organism present.

It is preferred to use a chemiluminescent material and then detect thepresence of the chemical light by a photograph or a digital imager suchas the MicroStar™ system available from Millipore Corporation ofBedford, Mass.

FIG. 2 shows another preferred embodiment of the present invention. Tothe extent that the reference numbers indicate the same steps as in FIG.1, the reference numbers have remained the same.

In this embodiment, after the enumeration and PNA probe steps have beencompleted, the results of each test is compared with each other todetermine whether there are indications of colonies of organisms on onetest in a certain location which are not found in the same location withthe other test. This allows one to eliminate any false positives fromthe results. This is particularly true with the PNA probe test whichsometimes identifies a location as containing a colony of an organismwhen further analysis indicates that no colony is present at thatlocation. The simplest means for doing this is use a MicroStar™ systemavailable from Millipore Corporation of Bedford, Mass. and tosuperimpose or view in parallel the two images. Alternatively, one canuse a software program to automatically compare the two test results anddiscount any spot not found in the same location on both tests.

This is a particularly useful in applications where the enumeration andidentification of microorganisms is required or desired.

EXAMPLE

Tests were conducted to determine whether the present process could beused to enumerate and distinguish two closely related microorganisms,Escherichia coli and Salmonella choleraesuis.

Pure cultures of Escherichia coli and Salmonella choleraesuis werediluted to an estimated 50 colony forming units per 100 μl in a sterile0.85% saline solution. Some of the two cultures were mixed together andfiltered as a mixture while the rest were filtered separately throughMicroStar™ filter discs in a glass filter holder, both available fromMillipore Corporation of Bedford, Mass.

The membranes were then incubated for six hours. The membranes from eachset (mixed as well as the individual organisms) were subjected to anATP-bioluminescence enumeration assay using the MicroStar™ Reagent KitRMLL00000 available from Millipore Corporation of Bedford, Mass. todetermine the number of colonies on each membrane. Samples of each setwere also stored on the traditional media growth plates and incubatedfor overnight at 30° C.

The same membranes were then subjected to a PNA probe assay using probesE. coli-specific Eco06™ probes available from Boston Probes, Inc. ofBedford, Mass.) and to a PNA probe assay for determining all bacteriaincluding the two used in this test (BacUni™ probes available fromBoston Probes, Inc. of Bedford, Mass.).

On the membranes where the blend of organisms were filtered onto thesame membrane, the number of colonies detected using the PNA probespecific for E. coli equaled the number of E. coli cells filtered, whilethe colonies observed with the universal bacteria probe equaled the sumof both organisms present.

In contrast, equivalent numbers of colonies were detected on themembranes that filtered only the E. coli, using either probe. Themembranes that filtered only the S. choleraesuis could only be detectedusing the universal probe.

Additionally, the organisms detected by the PNA probes were matched tothe locations found by the ATP-bioluminescence assay so as to eliminateany false positives caused by the membrane or its contamination.

These results were consistent with the numbers of colonies detected bythe ATP assays or on the growth control plates. This demonstrates that aspecific microbial species can be detected and enumerated using acombination of ATP and PNA probe assays on the same membrane.

1. A process for enumerating and identifying microorganisms comprising:(a) filtering a liquid sample through a membrane suitable for theretention of microorganisms, (b) incubating said membrane with anymicroorganisms, (c) applying an enumeration test to the surface of saidmembrane to enumerate the colonies of microorganisms that are present,and counting the number present, (d) applying a PNA hybridization to themicroorganisms on said membrane to determine the presence/absence of oneor more specified microorganisms, and counting the number detected, and(e) matching the organisms found by said enumeration test to thelocations of the colonies of organisms found by said PNA hybridizationstep and eliminating those locations found by the PNA hybridizationwhich do not correspond to the locations found by the enumeration test.2. The process of claim 1 wherein the liquid sample is from 50 to 1000milliliters, the membrane is selected from the group consisting of PVDFmembrane having hydrophilic areas separated by hydrophobic partitions,the membranes are incubated for 1 to 24 hours at a temperature of fromabout 24° C. to about 40° C. and the enumeration test is selected fromthe group consisting of an ATP-bioluminescence test and a fluorescenttag absorption test.
 3. The process of claim 1 wherein the enumerationtest is an ATP/bioluminescence test and is read by a digital imagingsystem.
 4. The process of claim 1 wherein the PNA hybridization step isread by a process selected from the group consisting of X-ray film,instant film, visual detection of a colorimetric reaction on themembrane, visual detection of a fluorescent tag or by digital imaging.5. A process for enumerating and identifying microorganisms comprising(a) filtering a liquid sample through a membrane suitable for theretention of microorganisms, (b) incubating the membrane with anymicroorganisms, (c) registering the membrane so as to indicate aspecific point along one edge of the membrane, (d) applying anenumeration test to the surface of the membrane to enumerate thecolonies of microorganisms which are present, and counting the numberpresent, (e) applying a PNA hybridization to the microorganisms on saidmembrane to determine the presence/absence of one or more specifiedmicroorganisms, and counting the number detected and (f) matching theorganisms found by the enumeration test to the locations of theorganisms found by the PNA hybridization through the use of theregistration mark and eliminating those locations found by the PNAhybridization which do not correspond to the locations found by theenumeration test.
 6. The process of claim 5 wherein the liquid sample isfrom 50 to 1000 milliliters, the membrane is selected from the groupconsisting of PVDF membrane having hydrophilic areas separated byhydrophobic partitions and the membranes are incubated from about 1 toabout 24 hours at a temperature of from about 24° C. to about 40° C. 7.The process of claim 1 wherein the membranes are incubated from about 3to about 8 hours.
 8. The process of claim 5 wherein the membranes areincubated from about 3 to about 8 hours.
 9. The process of claim 5wherein the enumeration test is an ATP/bioluminescence step that is readby a digital imaging system.
 10. The process of claim 5 wherein the PNAhybridization is read by a process selected from the group consisting ofX-ray film, instant film, visual detection of a colorimetric reaction onthe membrane, visual detection of a fluorescent tag or by digitalimaging.
 11. The process of claim 5 wherein the enumeration test is afluorescent tag absorption test.